Fuidi herd management schema

ABSTRACT

The invention is a herd management schema based upon the inventor&#39;s analysis of the natural history of bovine infection due to  Mycobacterium avium  subspecies  paratuberculosis  (Map) and related genomic variants and upon the ability of two distinct Map ELISA tests to sequentially or in parallel determine prior and current Map infection and evidence of active  mycobacterium  replication. Interpretation of the test results are integrated into sequential directives designed to enhance productive retention of infected animals as well as identify animal not previously infected. The sequential utilization of the data guidelines is developed to minimize the adverse economic impact.

FIELD OF INVENTION

The present invention is a business method/utility patent thatintegrates two types of serum ELISA tests for Mycobacterium aviumsubspecies paratuberculosis (Map) into a herd management schema thatcapitalizes on the natural history of Map infection.

DESCRIPTION OF PRIOR ART

The Disease

National Policy: Mycobacterium avium subspecies paratuberculosis (Map)is the causative agent of a chronic progressive granulomatous disease ofthe gastrointestinal tract, Johne's disease (Clarke 1997; Whitlock1996). The organism is global in its distribution and causes disease inboth wild and domestic herbivores. The granulomatous inflammation leadsto diarrhea, cachexia, and eventual death (Clarke 1997; Raizman 2005).Infection with Map is increasing in prevalence among food-producinganimal globally.

Economic Impact for the Dairy Industry: Estimates of herd prevalenceusing serological screening are comparatively low compared to thenumbers achieved with fecal culture screening. Within large herds, up to40% of cows may be infected (Chiodini 1983). Annual financial loss tothe United States Dairy Industry alone is estimated at $220 million(Otts 1999).

Zoonotic Potential: Animal disease control programs have tended to focuson diseases with major economic impact, trade implication and/orzoonotic potential (Grant 2005). The zoonotic potential of Map may soonover shadows its annual adverse economical impact. In its progressionfrom sub-clinical infection to disease, Map is introduced into the humanfood supply by virtue of its presence in milk. Map within milk is notadequately destroyed by pasteurization (Ayele 2005; Ellingson 2005;Giese 2000; Grant 2002; Miller 1996; Wuhib 2005).

Mycobacterium bovis is a documented zoonosis (Chiodini 1996;Herman-Taylor 1998). The organism is believed to pass from infectedanimals to man, primarily through oral ingestion of milk. The criticalquestion regarding Map is not whether the organism is a zoonosis, butrather, in its dissemination to humans is Map etiologically linked toCrohn's disease.

A strong circumstantial case is developing which suggests that Map canfunction as a zoonotic pathogen with respect to Crohn's disease.(Chiodini 1996; Mishina 1996; Naser 2000; Sechi 2001; Whittington 2000).Naser et al. isolated Map from the milk of two lactating women withCrohn's disease and none from five samples from normal control (Naser2004).

REFERENCES CITED

Ayele W. Y., Svastova P., Roubal P., Bartos M, Pavlik I.: Mycobacteriumavium subspecies paratuberculosis cultured from locally and commerciallypasteurized cow's milk in the Czech Republic. Appl. Envir. Microbiol.71:1210-1214, 2005

Clarke C. J. The pathology and pathogenesis of paratuberculosis inruminants and other species. J. Comp. Pathol. 116:217-261, 1997

Chiodini R. J., Van Kruiningen H. J., Merkal R. S.: Ruminantparatuberculosis (Johne's disease): the current status and futureprospects. Cornell Vet. 74:218-262, 1984

Chiodini R. J., Rossiter C. A.: Paratuberculosis: A potential zoonosis.:Vet. Clin. North America: Food Animal Practices 12:457-467, 1996

Ellingson J. L., Anderson J. L., Koziczkowski J. J., Radcliff R. P.,Sloan S. J., Allen S. E., Sullivan N. M.: Detection of viableMycobacterium avium subspecies paratuberculosis in retail pasteurizedwhole milk by two culture methods and PCR. J. Food Prot. 68:966-972,2005

Giese S. B., Ahrens P.: Detection of Mycobaterium avium subsp.paratuberculosis in milk from clinically affected cows by PCR andculture. Vet. Microbiol. 77:291-297, 2000

Grant I. R., Ball H. J., Rowe M. T.: Incidence of Mycobacteriumparatuberculosis in bulk raw and commercially pasteurized milk fromapproved dairy processing establishments in the United Kingdom. Appl.Envir. Microbiol. 68:2428-2435, 2002

Grant I. R.:Zoonotic potential of Mycobaterium avium subsp.paratuberculosis: the current problem. J. Appl. Microbiol. 98:1292-1290,2005

Hermon-Taylor J., Barnes N., Clarke C., Finlayson C.: Grand RoundsMycobacterium paratuberculosis cervical lymphadenitis, followed fiveyears later by terminal ileitis similar to Crohn's disease. British Med.J.316b:449-453, 1998

Millar D., Ford J., Sanderson J., Withey S., Tizard M., Doran T.,Hermon-Taylor J.: IS900 PCR to detect Mycobaterium avium subspeciesparatuberculosis in retail supplies of whole pasteurized milk in Englandand Wales. Appl. Environ Microbiol. 62:3446-52, 1996

Mishina D., Katsel P., Brown S. T. Gilberts C. A. M., Greenstein R. J.:On the etiology of Crohn's disease. Proc. Natl. Acad. Sci. USA93:9816-20, 1996

Naser S. A., Schwartz D., Shafran I.: Isolation of Mycobaterium aviumsubsp. paratuberculosis from breast milk of Crohn's disease patients.Am. J. Gastroentereol; 95:1094-1095, 2000

Naser S. A., Ghobrial G., Pomero C., Valentine J. F.: Culture ofMycobacterium avium subspecies paratuberculosis from the blood ofpatients with Crohn's disease. Lancet 364:1039-1044, 2004

Ott S. L., Wells S. J., Wagner B. A.: Herd-level economic lossesassociated with Johne's disease on US dairy operations. Prev. Vet. Med.40:179-192, 1999

Raizman E. A., Wells S. J., Jordan P. A., DelGiudice G. D., Bey R. R.:Mycobaterium avium subsp. paratuberculosis from free-ranging deer andrabbits surrounding Minnesota dairy herds. Can. J. Vet. Res. 69:32-38,2005

Sechi L. A., Mura M., Tanda F. Amelia L, Antonello S, Fadda G, andZanetti S.: Identification of Mycobaterium avium subsp. paratuberculosisin biopsy specimens from patents with Crohn's disease. J. Clin.Microbiol. 39:4514-17, 2001

Sechi L. A, Scanu A. M, Molicotti P, Cannas S, Mura M. Dettori G, FaddaG, Zanetti S.: Detection and isolation of Mycobaterium avium subspeciesparatuberculosis from intestinal biopsies of patients with and withoutCrohn's disease in Sardinia. Am. J. Gastroenteriol. 100:1529-1536, 2005

Whitlock R. H., Buergelt C. D.:Preclinical and clinical manifestationsof paratuberculosis (including pathology). Vet. Clin. N. Am. 12:357-371,1996

Whittington R J, Hope A F, Marshall A. D.: Molecular epidemiology ofMycobaterium avium subsp. paratuberculosis: IS900 restriction fragmentlength polymorphism and IS 1311 polymorphism analysis from animals and ahuman in Australia J. Clin. Microbiol 38:3240-3248, 2000

Wuhib Y. A., Svastova P., Roubal P., Artos M., Paylik I.: Mycobacteriumavium subspecies paratuberculosis cultured from locally and commerciallypasteurized cow's milk in the Czech Republic. Appl. Environ Microbiol.71:1210-1214, 2005

Serum ELISA Mycobacterium Avium Subspecies Paratuberculosis Tests

ELISA Test Sensitivity

Two Map ELISA tests are in current use in the United States. Thecommercial Map ELISA tests, ParaChek® (Prionics, Zurich, Switzerland)and HerdChek® (IDEXX Laboratories Inc. Westbrook, Me.) are based on thelipoarabinomannan polysaccharide (LAM) or other Map membrane relatedconstituents. The Map strains used for diagnostic testing posses the IS900 DNA insertion sequence. Map polymerase chain reaction (PCR) testsusing primers derived from the IS900 gene sequence are used to conferspecificity to suspected culture isolates. This insertion sequence isthe principle means by which Map is differentiated from othermycobacteria (Sugden, Harris).

While there is a high degree of correlation between both types ofcommercial tests when done on clinically ill (fecal shedding, weightloss and diarrhea) animals, both types of Map ELISA tests fail toidentify a significant number of fecal culture positive animals. Thetests are used to identify negative, suspicious, and positive isolates.In terms of developing overt clinical disease, a sero-positive isolatehas a positive predictive value of 74. A positive commercial Map ELISAtest and/or fecal culture evidence of significant fecal shedding are thecriteria (USDA's advocated policy of test-and-cull) in current use bywhich a decision is reach to remove a given animal from the herd(Harris). No diagnostic significance has been given to transientnon-diagnostic values identified by either of the commercial Map ELISAtests. Despite the use of test-and-cull schema in herd management, theprevalence of Map in dairy herds has progressively increased.Sereologically positive, subclinically infected, as well as diseaseddairy cows have been shown to shed Map into their milk (see enclosedpaper) and potential introduce zoonotic pathogens into the human foodchain.

Collins et al. evaluated five antibody detection tests for the diagnosisof bovine paratuberculosis using serum samples from 359 dairy cattle inseven paratuberculosis-free herds and 2,094 dairy cattle in sevenMap-infected dairy herds. Both the ParaChek® and HerdChek® (IDEXX) ELISAtests done in accordance with manufacturers' instruction and interpretedas prescribed by the kit insert, identified less than 29% of fecalculture positive cows. Linear regression analysis of quantitativeresults showed a low correlation co-efficiency. Sockett et al. reportedthe sensitivity of commercial Elisa's for cattle to be 8.9 to 32.1% forlow shedders and 47.1 to 62.9% for midlevel shedders. Sweeney et al.have suggested that commercial Elisa's might have a sensitivity ratelower than 13.5%. McKenna et al. tested sera collected from dairy cowsat slaughter in assessing the agreement with documented infection ofthree commercially available Map ELISA tests which included HerdChek®and ParaChek®. The investigators found a poor agreement between thethree ELISA tests and infected cows. Mycobacteria have been shown to bemore readily identified within feces when the currently commerciallyavailable Map ELISA tests are in their projected positive diagnosticzones (Cocito). The high incidence of false negatives with the ParaChek®and/or HerdChek® ELISA tests has limited utility to identifying animalswith an advanced disease state. A second concern with the membrane-basedMap ELISA tests is the occurrence of false-positive test results.

Over all, the ParaChek® and HerdChek® Map ELISA tests suffer from a lackof sensitivity in identifying animals with positive fecal cultures. Mostclaims of sensitivity have focused upon heavy shedding and/or clinicallydiseased animals. A suspicious or positive test did not necessarilypredict outcome. The quest for antigenic specificity excluded the issueof genetic polymorphism within Mycobaterium avium subsp.paratuberculosis (Map).

A second type of Map ELISA test was develop utilizing primarilyprotoplasm from a Mycobacterium 18 that was initially though to be Mapbut was later demonstrated to be more Mycobaterium avium than Map.Current data referable to the sensitivity of protoplasmic or wholeorganism antigen-based Map ELISA tests is lacking. While these testsidentify diseased animals, they also produced a significant number ofpositive test results in animals that lacked confirmation by fecalculture or longitudinal clinical assessment. This fact significantlylimited their utilization and virtual abandonment.

REFERENCES

Cocito C., Gilot P., Coene M. de Kesel M., Poupart P., Vannuffel P.:Paratuberculosis. Clin. Microbiol. Review 1994; 7:328-345

Collins M. T., Wells S. J, Petrini K. R. et al.: Evaluation of fiveantibody detection tests for the diagnosis of bovine paratuberculosis.Clin. Diagn. Immunol. 2005; 12: 685-692

Harris N. B., Bareletta R. G.:Mycobaterium avium subsp. paratuberculosisin Veterinary Medicine. Clin. Microbiol Reviews 2001;14:489-512

McKenna S. L. B., Barkema H. W., Keefe G. P., Sockett D. C.: Agreementbetween three Elisa's for Mycobaterium avium subsp. paratuberculosis indairy cattle. Vet. Microbiol. 2006; 31:285-291

Sockett D. C., Conrad T. A., Thomas C. B., Collins M. T.: Evaluation offour serological tests for bovine paratuberculosis. J. Clin. Microbiol.1992; 30:1134-1139

Sugden E. A., Stilwel K., Michaelides A.: A comparison oflipoarabinomannan with other antigens used in the absorbed enzymeimmunoassay for serological detection of cattle infected withMycobacterium paratuberculosis. J. Vet. Diagn. Invest. 1997;9:413-417

Sweeney R. W., Whitlock R. H., 12. McAdams S., Fyock T.: Longitudinalstudy of ELISA seroreactivity to Mycobaterium avium subspeciesparatuberculosis in infected cattle and culture-negative herd mates. J.Vet. Diagn. Invest. 2006;18:2-6

DESCRIPTION OF THE INTELLECTUAL BASIS OF THE INVENTION

The invention is based upon re-defining existing data concerning MapELISA tests in light of novel information derived by the inventor.

The existing information used was the antigenic base of the currentcommercial Map ELISA tests: the lipoarabinomannan polysaccharide (LAM)and or related lipoprotein membrane constituents. This information wascombined with the fact that these types of antigens tend to be ofantibodies that are of relatively short duration compared withantibodies elicited by ribosomes and nucleic acid constituents.

Collaborative research done with United States Department ofAgriculture' South Florida Dairy Herd Demonstration Project provideinsight that alter perception of the natural history of Map infection.In a study of Map ELISA titers derived over a 14 month period, it wasnoted that approximately 10-11% of dairy cows would develop transientlow level diagnostic antibodies which would disappear when retested. Theliterature infers the progressive nature of Map.

First, sixty-six sera were obtained from a North Florida dairy herdwhich had a high incidence of clinical disease. All tests were run inaccordance with the manufacturers' specification. Positive and negativecontrols were utilized. Seven sera were identified as being positive byall three tests: six by the ParaChek® test, six by the HerdChek® test,and seven by a protoplasmic -based ELISA test. The ParaChek® and IDEXXtests each failed to identify a positive sample identified by the othertest. The higher the protoplasmic-based ELISA test registered, thegreater the probability that the corresponding ParaChek® and/orHerdChek® would be positive. The protoplasmic antigen-based testidentified an additional 11 cows as having significant titer (14).

REFERENCE

Williams E., Monif G. R. G., Buergelt C. D.; Comparative analysis ofdifferent Map ELISA tests. The Paratuberculosis Newsletter. March2008:9-10

In an unpublished study, the FUIDI (a whole organism antigen basesource) and ParaChek® Map ELISA tests were compared in parallel on thesame serum specimen (Table 1-1). In Herd #1 that was reputedly Map-free,the ParaChek® ELISA test did not identify any serum specimens as beingpositive. The FUIDI ELISA test identified 10 animals as being infected.In Herd #2 that had a significant number of seroreactive cows, theParaChek® ELISA 2 as being positive and 10 as being suspicious. TheFUIDI ELISA test identified 16 as being positive and 3 as beingsuspicious.

This observation lead to the comparative used of a Map ELISA test whoseantibody capturing capacity was dictated by LAM and a modified Map ELISAtest (FUIDI) that identified antibodies to the protoplasmic massantigens as wells as surface membrane elements

Significance of Diverging Map ELISA Test Results in Dairy Cows Gilles R.G. Monif, Joseph E. William

Johne's disease is a chronic granulomatous disease of the bovinegastrointestinal tract caused by Mycobaterium avium subspeciesparatuberculosis (Map) (1). The adverse economical impact of diseaseglobally is estimated at 1.5 billion dollars annually (2).

The commercial Map ELISA tests have very poor sensitivity in identifyinginfected cows (3,4). Collins et al. evaluated five antibody detectiontests for the diagnosis of bovine paratuberculosis using serum samplesfrom 359 dairy cattle in seven paratuberculosis-free herds and 2,094dairy cattle in seven Map-infected dairy herds (5). Both the ParaChek®(Prionics, Zurich, Switzerland).) and HerdChek® (IDEXX Laboratories Inc.Westbrook, Me.) ELISA tests done in accordance with manufacturers'instruction and interpreted as prescribed by the kit insert, identifiedless than 29% of fecal culture positive cows. Linear regression analysisof quantitative results showed a low correlation co-efficiency. Sockettet al. reported the sensitivity of commercial Elisa's for cattle to be8.9 to 32.1% for low shedders and 47.1 to 62.9% for midlevel shedders(6). Sweeney et al. have suggested that commercial ELSAs might have asensitivity rate lower than 13.5% (7). McKenna et al. tested seracollected from dairy cows at slaughter in assessing the agreement withdocumented infection of three commercially available Map ELISA testswhich included HerdChek® and ParaChek®. The investigators found a pooragreement between the three ELISA tests and infected cows (8).Mycobacteria have been shown to be more readily identified within feceswhen the currently commercially available Map ELISA tests are in theirprojected positive diagnostic zones (9).

A prior comparison of the IDEXX®, ParaChek®, and FUIDI Map ELISA #1tests had shown excellent correlation between the three tests when thediagnostic criteria of either the IDEXX® or ParaChek® tests were used toestablish the denominator (12). Seven cows out of 66 cows wereidentified by all three tests: 6 by the ParaChek® test, 6 by the IDEXXtests and 7 by the FUIDI test. The IDEXX® and ParaChek® tests eachfailed to identify a positive serum identified by the other. The FUIDIMap ELISA test identified 11 additional dairy cows as being infectedwith Map. The higher the FUIDI titer, the better was the correlationbetween the three tests. That is discrepancy between the FUIDI andcommercial tests was not simply a case of over-diagnosis was inferredwhen the ParaChek® and FUIDI Map ELISA tests were test against 9 serafrom cows with necropsy confirmed Johne's Disease (13). In the ParaChek®Map ELISA test, four sera demonstrated any activity, but only one waspositive and one was suspicious. In the prototype FUIDI test, six werepositive. With the new diagnostic groupings for the FUIDI test, threewould have been positive and three would have fallen in the suspiciouscategory.

To achieve the mandated specificity indicative of only Map, the targetantigens of the current commercial Map ELISA tests have had to be basedupon a limit antigenic array such as the lipoarabinomannan or selectedMap surface proteins. The serological response is a partial function ofantigen complexity (10,11). Different antigens elicit divergent types ofantibodies. Whole organism antigenic utilization elicits an array ofantibodies whose spectrum of reactivity exceeds that induce by subunitsof the organism. By using a modified whole organism, the FUIDI testpresents a significantly broader antigen array.

The purpose of this study is to analyze the possible significance ofidentification of any Map antibody titer by the ParaChek® Map ELISA testin conjunction with the corresponding FUIDI #1 Map titer and comment onthe ramification derived from both tests as they apply to the naturalhistory of bovine Map infection as opposed to Map disease.

Materials and Methods

Study Population:

Infectious Disease Incorporated (IDI) through its strategic partnershipwith the University of Florida. College of Veterinary Medicine (UFCVM)was given access to sera and serological data derived from the 2008USDA's Florida Johne's Disease Dairy Herd Demonstration Project (14).The study sera were determined a given cow's serum having been tested bythe State of Florida Diagnostic Laboratory in December of 2006 as wellas February 2008 and its subsequent availability for retesting in 2008.The ParaChek® and FUIDI ELISA test results were derived by testing thesame February 2008 serum samples.

ParaChek Map ELISA Test: The ParaChek® Map ELISA tests were done at theState of Florida Diagnostic Laboratory-Live Oak in accordance to themanufacturer's instructions. Positive and negative controls wereutilized. The test results were forwarded to the USDA's Office inGainesville Florida. A suspicious reading was 0.50 to 0.99. A readingequal to or above one (1) was deemed positive.

Analysis of the ParaChek® Map ELISA data has presumed that the evidencepresented to U.S. Department of Agriculture for commercial acceptanceand certification had to have convincingly demonstrated that the testtruly identifies antibodies specific to Map.

FUIDI Map ELISA#1 Test:

The FUIDI Map ELISA test #1 uses primarily a modified antigen arraybased primarily on a Map isolate originally thought to be Map, but laterbeen shown to be more M. aviurm-like. Test sera were pre-absorbed withMycobacterium pheli. ELISA results were calculated from absorbance at OD405 nm. Readings less than 1.9 optical density (OD) were deemednegative; readings between 1.9 and 2.4 were deemed as requiring closemonitoring/suspicious. Readings of 2.5 OD or greater were calledpositive. All FUIDI Map ELISA #1 tests done at UFCVM were run intriplicate with both positive and negative controls. The test resultswere forwarded to the USDA's Office in Gainesville Florida.

Fecal Cultures: Fecal samples were shipped FedEx in coolers with icepacks to the Animal Disease Diagnostic Laboratory at Purdue Universityand processed according to that laboratory's internal protocols.

Data Processing:

The ParaChek® ELISA test results from the 2007 Florida Johne's DiseaseDairy Herd Demonstration Project (testing done Dec. 13, 2006) were madeavailable during the study. Once the final FUIDI Map test results hadbeen submitted to USDA, the 2008 Florida Johne's Disease Dairy HerdDemonstration Project test results were forwarded for comparativeanalysis.

Statistical Analysis:

The random selection of the study sera which lacked specific targetedsubgroups limited meaningful statistical analysis. For relativecomparisons only, likelihood ratios (LR) were constructed for the FUIDIMap ELISA data to lend possible insight as to whether or not a FUIDIpositive test result would predict a previously or concurrently Mapdairy cow with Map specific antibodies. The LRs were calculated asfollows: LR=(number of sera with a positive FUIDI Map ELISA test resultdivided by the number of sera which over approximately 14 months hadbeen shown to have Map specific antibody; the number of positive andsuspicious FUIDI ELISA tests divided by the number of ParaChek® serawhich identified newly developed Map antibodies; the number ofFUIDI-positive sera divided by the number of ParaChek® sera with Mapantibodies at any time.

Results:

Of the 140 sera tested in February of 2008, 103 had been tested onDecember of 2006. In December of 2006, 10 of the 103 sera test had anytiter as measured by the ParaChek® Map ELISA test. Two wereParaChek®-positive. One serum was identified as beingsuspicious/inconclusive.

When reanalyzed in 2008, no data was available on one of theParaChek®-positive animals (#2920; however, a strong positive titer wasrecorded in the FUIDI test (Table 2). One low ParaChek® tittered cow(#1011) had its ELISA test values moved into from 0.08 into the test'spositive zone (2.38). Five previously low tittered sera became totallynegative.

Two cows had ParaChek® reading in the test's positive range. Nofollow-up ParaChek® test was done for cow #2920; however the highpositive FUIDI reading is consistent with a continued high level ofantigen processing. The other ParaChek® positive cow (#2290 exhibited avery significant drop in demonstrable Map antibodies (1.32 to 0.050. Thesuspicious ParaChek® serum similarly fell out of its diagnostic zone(0.72-0.02).

The remaining cow's serum maintained a persistent low Map antibodypresence. Of these 11 sera which had any Map antibodies by the ParaChek®Map ELISA, 4 were positive and 3 were suspicious as determined by theFUIDI Map ELISA. The LR for the sera deemed positive in the FUIDI ELISAtest is 0.40. If the suspicious and positive FUIDI results are combined,the FUIDI ELISA test's LR becomes 0.70

Nine out of the 93 cows without prior Map antibodies in December 2006developed low level of Map antibodies when tested in February of 2008(Table 3). The corresponding FUIDI tests were positive in two cases andsuspicious in two others. The combined FUIDI positive and suspiciousresults create a LR of 0.56.

Table 4 reflects the shift of emphasis from ParaChek® directedcomparisons to FUIDI directed comparisons. The FUIDI Map ELISA#1 testwas positive in 12 instances. The presence of prior or concurrent Mapantibodies as demonstrated by the ParaChek® Map ELISA test wasidentified in 8 instances. Of the 21 animals postulated as requiringadded surveillance/monitoring (suspicious category), six had prior orcurrent identification of specific Map antibodies as identified by theParaChek® ELISA and 4 of the 21 cows had a positive fecal culture.Combining the two criteria, prior demonstration of Map specificantibodies by the ParaChek® test and positive fecal cultures,approximately ⅓ of the cows in the FUIDI suspicious category hadevidence of Map infection.

Two cows out of the 37 cows not previously tested in December of 2008developed low level Map antibody titer as determined by the ParaChek®test. For one of the two cows, the corresponding FUIDI test waspositive.

Eleven cows had Map recovered from a fecal specimen (Table 5). Of the 11Map positive fecal cultures, the ParaChek® test identified one as beingpositive in February 2008 (LR =0.09) The FUIDI identified in 2008 one asbeing positive and four as being suspicious (combined LR =0.36).

Discussion:

The primary focus of the comparison of the ParaChek® and FUIDI MapELISA#1 tests was not to document that one test is necessarily betterthan the other, but rather to point out that these two ELISA tests donot necessarily convey the same information applicable to the naturalhistory of bovine Map infection. When the 2008 suspicious and positiveFUIDI Map ELISA#1 test results are compared with sera that, in either2007 or 2008, a sufficiently high correlation can be shown supportingthe contention that the cows so identified as having any Map antibodytiter in the ParaChek® test have experience infection with Map. Theoverlapping correlation over time between a positive FUIDI ELISA titerand any titer ParaChek® ELISA test result strengthens the contentionthat any antibodies identified by the ParaChek® Map ELISA test arespecific Map antibodies.

The FUIDI Map ELISA#1 was selected for development and testingpredicated upon its ability to identify Map-like mycobacteria as well asMap. The rational for this decision was the contention that Maps strainsevolved from a Mycobacteria avium base and its genomic differentiationhas resulted in genomic polymorphism of mycobacterium stains that arecapable of inducing Johne's disease (14-24).

The FUIDI Map ELISA#1 test results can be challenged by the contentionthat they also identify environmental mycobacteria. Osterstock et al.parenterally immunized beef calves with Mycobacterium celatinum, M.scrofulaceum, M. intracellulare, and M. avium subspecies avium (25).False-positive ELISA tests were recorded with both commercial Map ELISAtests currently used in the United States. The authors contended thatenvironment mycobacteria were responsible for some of the false positivetests previously observed in beef cattle. What they more probablydemonstrated IS900 insertion sequence may share antigenic elements withselected environmental mycobacteria. The production of ELISA-positiveantibodies directed against an environmental mycobacterium had requiredthe subcutaneous injection with adjuvant of large numbers of organisms.In order to achieve a comparable ParaChek®-positive antibody response innature would require organism internalization, antigen processing, and alarge antigen mass: more specifically defined, infection.

In contrast to intracellular mycobacteria, the vast majority ofpathogenic bacteria undergo complete organism elimination. In the courseof disease, extremely high titers of antibodies directed againstlipopolysaccharide and lipoprotein membrane components are produced;however their detection is relatively short-lived. The explanationadvanced to account for disappear of specific antibody titer has beenthat the eliciting antigenic templates are no longer available (27,28).

The appearance and disappearance of ParaChek® identified Map antibodiesappears to be a direct function of the elicitor antigenic component usedto develop the test. The ParaChek® Map ELISA test appears to becontingent upon continued processing of its antigenic elicitor forantibody production.

The loss of ParaChek® Map detectable antibodies implies that the host'simmune response has achieved immunological governance. Mycobacteria,like M. tuberculosis, despite recovery, do not undergo completeelimination from the body. Evidence of an immune response persists yearsafter the initial system introduction of the organism into the hostanimal (26). Reactivation of organism replication can occur when thehost animal's immune system has undergone significant compromise (26).

By itself, the interpretation of data implying immune governance couldbe open to debate. What the FUIDI Map ELISA#1 data does is lendsubstantial credence to the contention that these animals which havelost ParaChek® titer have had or are experiencing on a non-detectablelevel by the ParaChek® test infection with Map.

Epidemiologically, the small fragments of data relative to the loss ofantibody titer directed against surface component of Map imply that anumber of dairy cows infected with Map may achieve immunologicalgovernance. Fifty percent of the 10 cows with any Map antibodies hadtheir titer in the ParaChek® test revert to a non-detectable titer uponretesting. Expansion to observations to an analysis of 602 dairy cowsfrom which the 103 study sera were derived reveals that 46 of the 62cows (75.8%) with previously identified Map antibodies lost all titerupon retesting.

A significant bias in the ParaChek® Map antibody group was the relativeabsence of animal identified by testing as being suspicious or positive.The herd's management schema dictated that serologically positive orsuspicious cows, cows with significant fecal shedding, and/or cows withclinical signs be systematically culled from the herd. Nevertheless,three such animals were present in the study population: 2 testParaChek®-positive and 1 ParaChek®-suspicious. Of the twoParaChek®-positive cows, one is inferred to have had a high level ofantigen processing by the corresponding high positive FUIDI titer. Theother positive cow had her ParaChek® titer drop from 1.32 to 0.05.Similarly, the suspicious cow's ParaChek® titer fell from 0.72 to 0.02.In both cases, the corresponding FUIDI titer was non-diagnostic.

The FUIDI Map ELISA#1 test appears to better identify the probability ofeither significant prior or current Map infection. The FUIDI antigenicbase is constituted by a broad array immunogens, some of which aretheorized to persist along with evidence of specific cell-mediatedimmunity. Of the 10 sera with any positive antibody titer as determinedby the ParaChek® done in December 2006, the FUIDI ELISA test waspositive for 4 and suspicious for another 3, giving a combined LR of0.7. Nine previously negative ParaChek® sera developed low level of Mapspecific antibodies. The corresponding FUIDI titer was positive in 2cases and suspicious in 2 of the 9 cases giving the combined group a LRof 0.56.

Of the 17 positive sera identified by the FUISI Map ELISA#1 test, 6identified cows that had or had had evidence as specific Map antibodiesas determined by the ParaChek® Map ELISA test (LR=0.35). If corrected toinclude a cow with positive fecal cultures the LR becomes 0.40.

Of the 21 suspicious FUIDI sera, 6 cows had or had had evidence of Mapantibodies as determined by the ParaChek® Map ELISA test (LR=0.29). Twocows with no detectable Map antibodies, but suspicious in the FUIDI testwere present.

For the cows with no detectable Map antibodies in 2007 and 2008, thereis no way of accessing if in years prior to 2007 that they had notacquired Map infection with subsequent acquisition of immune governancewhich may account for some of the false positive FUIDI titers recorded.

The ParaChek® Map test appears to identify ongoing Map membrane antigenprocessing. The magnitude of the titer indicates the probability offuture disease induction; however a diagnostic test does not necessarilypredict future outcome. One ParaChek®-positive cow and one cow with asuspicious titer in 2007 when retested had insignificant residual titerin 2008.

From the Map ELISA test comparisons, any demonstration of Map specificantibodies by the Parachek® Map ELISA test indicates systemic antigenicprocessing. The FUIDI-positive Map ELISA#1 test appears to identify bothunderlying as well as current Map infection. Like the ParaChek® testdata, the FUIDI #1 test results can not predict outcome, justprobability. The FUIDI Map ELISA#1 test's utility appears lie in itsability to better identify a subpopulation that could profit from moreintensive monitoring.

From a parallel use of both ELISA tests, a better perception of thenatural history of Map bovine infection is likely to emerge.

REFERENCES

1. Stabel J. R.: Johne's disease: a hidden threat. J. Dairy Science1998; 81:283-288

2. Chi J., Van Leeuwen J. A., Weersink A., Keefe G. P.: Directproduction loses and treatment costs from bovine viral diarrhea virus,bovine leucosis virus Mycobaterium avium subsp. paratuberculosis andNeospora caninum. Prevent. Vet. Med. 2002; 69:2075-2082

3. Eamens G J, Whittington R J, Marsh I B, Turner M J, Saunders V,Kemsley P D, Rayward D: Comparative sensitivity of various fecal culturemethods and ELISA in dairy cattle herds with endemic Johne's disease.Vet. Microbiol. 2000; 77:357-367

4. Sweeney R W, Whitlock R H, Buckley C L, Spencer P A: Evaluation of acommercial-linked enzyme immunosorbent assay for the diagnosis ofparatuberculosis. J. Vet. Diagn. Invest. 1995; 7:488-493

5. Collins M. T., Wells S. J, Petrini K. R. et al.: Evaluation of fiveantibody detection tests for the diagnosis of bovine paratuberculosis.Clin. Diagn. Immunol. 2005; 12: 685-692

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8. McKenna S. L. B., Barkema H. W., Keefe G. P., Sockett D. C.:Agreement between three Elisa's for Mycobaterium avium subsp.paratuberculosis in dairy cattle. Vet. Microbiol. 2006; 31:285-291

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11. Collins M. T., Gardner I. A., Garry F. B., Roussel A. J., Wells S.J.;Consensus recommendations on diagnostic testing for the detection ofparatuberculosis in cattle in the United States. J. Am. Vet. Med. Assoc.2006; 229:19121918

12. Williams E., Monif, G. R. G., Buergelt C. D.: Comparative analysisof different Map ELISA tests The Paratuberculosis Newsletter March2008:7

13. Williams E., Monif G. R. G.: Comparative Map ELISA tests done oncows with necropsy documented disease. The Paratuberculosis Newsletter.March 2008: 7-8

14. Harrell C. D., Gauthier J., Berman W.: Unpublished data.

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16. Whittington R., Marsh I., Chow E., Cousins D.: Polymorphism inIS1311, an insertion sequence common to Mycobaterium avium subsp.paratuberculosis, can be used to distinguish between and within thesespecies. Mol. Cell. Probes 1998;12: 349-358 17. England S., Bolske G.,Johnansson: An IS900-like sequence found in Mycobacterium sp. other thanMycobacterium avium subspecies paratuberculosis. FEMS Microbiol. Lett.2002; 34:734-737

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20. Cousins D. V., Whittington R., Marsh I. Masters R. J., Evans R. J.,Kluver P.: Mycobacteria distinct from Mycobaterium avium subspeciesparatuberculosis isolated from feces of ruminants posses IS900-likesequences detectable by polymerase chain reaction: implications fordiagnosis. Mol. Cell. Probes1999; 14:431-442.

21. Coffin J. W. C., Condon C. A., Compston K. N. et al: Use ofrestriction fragment length polymorphisms resolved by pulsed-field gelelectrophoresis for subspecies identification of mycobacteria in theMycobacterium avium-complex and for isolation of DNA probes. J. Clin.Microbiol.1992; 30:1829-1836.

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What is established from the cited data is that the transient positivetiters identified by a LAM-based ELISA test were indicative of their Mapspecific antibodies. Their disappearance inferred that like, the humanmodel, Mycobacterium tuberculosis, infection and immune capture arecommon phenomenon and progression of infection to Johne's disease is arelative rarity.

The positive titers identified by protoplasmic antigen based tests arenot false-positives but, with reasonable probability, identified priorMap infection (comparable to a positive PPD reaction to tuberculin), andthe so-called false-positive tests observed with LAM-based Map ELISAtests are animals which subsequently achieved immune capture of theorganism.

The novelty of the invention is how these new observations are used tocreate a comprehensive herd management schema which will allow domesticanimal producers to better manage Map within their herd by retaininginfected animals which by other criteria would be culled.

Prior to the development of the FUIDI Herd Management Schema, the goldstandard was annual serological testing of animals using either theParaChek® or HerdChek® Map ELISA tests and/or fecal culture to determineif an animal should be removed from a herd for production considerationsand/or for constituting a greater threat of introducing Map into thehuman food chain. The guidelines used in USDA's South Florida Dairy HerdDemonstration Program were

-   -   Serological surveillance with a LAM-based Map ELISA test,    -   Fecal culture (elimination of significant shedders),    -   Clinical impression of disease, and    -   Significant decrease in milk and milk fat production.

This advocated approach is diseased focused and does not effectively usethe now perceived natural history of Map infection nor the utility ofdual surveillance within a Herd Management Schema to better identifyinfection with Map and determine the significance of a serological Maptiter.

The FUIDI Herd Management Schema utilizes

-   -   a serological single or multi-organism prototoplasm-based test        to identify both current and past Map infection,    -   a LAM-based Map ELIISA test to determine any degree of Map        replication,    -   the fact that the majority of infected animals with low level        and suspicious levels Map antibodies will achieve subsequent        immune capture to a varying degree,    -   the fact that 20-25% of animals with a diagnostic level of Map        antibodies may also achieve subsequent immune capture,    -   the fact that animals with recent immune capture at the time of        parturition may experience reactivation of infection,    -   The fact that majority of animals who are low Map fecal shedders        and are serologically negative will exhibit subsequent immune        capture.

The novelty in the information and its sequential utilization constitutethe basis for the invention and its claim to being a business methodutility patent.

FIG. 1—Part I of the FUIDI Herd Management Schema schematically presentsthe sequence of events when a diagnostic serum sample is submitted to aFUIDI certified veterinary diagnostic laboratory. The FUIDI testinvolves two Map ELISA tests that differ in their antigen spectrum andnot completely overlap. Both FUID#1 and FUIDI#2 tests are advocate, butcost considerations make sequential use more likely to be chosen withserum reanalysis with the FUID#2 test indicate only when FUIDI#1 hasindicated serological evidence of current or prior significant B-cellstimulation. A negative Map FUIDI#1 test indicates that no evidence ofMap directed B-cell stimulation is detected and the action advocated isto retest the animal annually.

If the FUIDI#1 test is suspicious or positive, the serum sample isretested to assess the probability of active antigen processing. If theFUIDI#2 test is negative, the recommended action is to retest the animalannually and/or after calving.

If a non-diagnostic or low titer is identified by the FUIDI#2 Map ELISAtest, the interpretation is that a low-level ongoing infection ispresent. The vast majority of these animals will achieve immunologicalcontainment of mycobacterium replication. To assess, whether immunecontainment or progression towards disease is likely to ensue, therecommended action is to retest the animal using the FUIDI#1 and #2 MapELISA tests in 3 months. If a diagnostic titer is identified, theinterpretation is of high level mycobacterium replication. While theanimal has about a 1 in 4 chance of achieving immunological containment,such an animal is, more likely than not a high fecal shedder of Map andshould be culled. If a valuable animal or clinical parameter such asbody score and milk/fat production are good, quantitative fecalculturing may provide valuable information for the information decisionmaking.

FIG. 2—Part II of the FUIDI Herd Management Schema. Part II is designedto provide an additional level of monitoring to determine theprobability of immunological containment versus progression of infectiontowards disease for a given animal. Fecal culturing of animals with highdiagnostic FUIDI Test #2 results is reserved for uniquely valuableanimals (bulls, valuable zoo animals) that have been isolated and forwhom enhancement of their immune system is being implemented.

A fecal specimen is obtained for quantity assessment by direct fecalculture or by direct fecal nested or real-time Map polymerase chainreaction (PCR) tests A negative culture indicates either immunologicalcontainment or sample error. The more probable demonstration indicatesnon-concurrence between serological data and organism shedding andwarrants retesting of subsequent sera using FUIDI#2 Map ELISA test tosee if serum titers decrease. If heavy fecal shedding is confirmed, therecommended action is to cull.

FIG. 3—Part III of the FUIDI Herd Management Schema focuses on animalsthat have had inferred active low level infection as determined theFUIDI #2 Map ELISA test. Production salvage of infected animals withinthis group is a key feature of the FUIDI Herd Management Schema.

The key indicator as to prognosis is the diagnostic readings obtainedupon serial FUID#2 testing. If the titer disappears or dropssignificantly, the potential for having Map into the milk, other than bycontamination, is markedly reduced. If the FUID#2 titer remainsrelatively comparable, unless the animals immune system is up regulated,the probability of progression to disease and Map shedding into aretheoretically enhanced. If the titer increase by 0.4 or greater, eventhough not in the test's diagnostic range, the recommended action is tocull.

It should be understood that the examples and embodiments describedherein in FIGS. 1-3 are for illustrative purposes and that variousmodifications or changes in light thereof will be suggested to personsskilled in the art and are to be included within the spirit and purviewof this application or embodiment thereof disclosed herein can becombined with any and/or all other elements or limitations (individuallyor in any combination) or any other invention or embodiment thereofdisclosed herein, and all such combinations are contemplated with thescope of the invention without limitations.

TABLE 1 Comparison of FUIDI and ParaChek ® Map ELISA tests Parachek ®Comparison Source Dairy Cow# FUIDI ParaChek 1* 8384 2.62 Neg Dairy 18661 2.61 Neg Dairy 1 8369 2.19 Neg Dairy 1 3721 2.46 Neg Dairy 1 35342.9 Neg Dairy 1 8291 1.94 Neg Dairy 1 8244 3.6 Neg Dairy 1 8064 2.08 NegDairy 1 8673 3.25 Neg Dairy 1 3119 2.11 Neg Dairy 1 7301 1.56 Neg Dairy1 3238 1.46 Neg Dairy 1 6279 2.73 Neg Dairy 1 3815 1.65 Neg Dairy 1 88001.41 Neg Dairy 1 3361 1.22 Neg Dairy 1 2916 0.96 Neg Dairy 1 3405 1.4Neg Dairy 1 7345 0.88 Neg Dairy 1 7659 0.58 Neg Dairy 1 3201 1.29 NegDairy 1 7519 0.63 Neg Dairy 1 3870 0.92 Neg Dairy 1 3520 0.65 Neg Dairy1 7304 0.82 Neg Dairy 1 7658 0.61 Neg Dairy 1 3737 1.24 Neg 2* 28 1.71Neg Dairy 2 93 4.5 Susp Dairy 2 125 2.79 Neg Dairy 2 306 2.43 Neg Dairy2 329 2.03 Susp Dairy 2 358 3.48 Neg Dairy 2 458 4.22 Susp Dairy 2 5871.41 Neg Dairy 2 293 1.02 Neg Dairy 2 699 2.4 Neg Dairy 2 734 2.19 SuspDairy 2 837 1.8 Susp Dairy 2 839 2.07 Susp Dairy 2 1260 2.67 Susp Dairy2 1273 3.37 Susp Dairy 2 1514 6.57 Pos Dairy 2 1625 2.2 Neg Dairy 2 17184.87 Susp Dairy 2 1733 2.92 Neg Dairy 2 1581 1.72 Neg Dairy 2 2020 1.4Neg Dairy 2 2075 0.95 Pos Dairy 2 1995 3.76 Susp Dairy Herd #1 -reputedly Map-free Dairy Herd #2 - known to have a significant # ofsero-reactive cows FUIDI values - .1.5 = negative; 1.51-1.99 =suspicious; 2.0 and above = positive ParaChek ® FUIDI SourcePositive/inconclusive positive/suspicious Dairy Herd #1 0/0  10/0 DairyHerd #2 2/10 16/3

TABLE 2 Comparison of ParaChek ® ELISA Titers February 2008 with FUIDIELISA Titers on Sera with Pre-existing Map Antibodies ParaChek ® Cow #Dec. 13, 2006 Feb. 28, 2008 FUIDI#1 1011 0.08 2.38 2.5 2290 1.32 0.051.0 2484 0.72 0.02 1.6 2920 1.42 n.d. 3.6 3125 0.02 0 3.3 3297 0.04 03.4 3728 0.08 0 2.1 3731 0.03 0 1.2 3739 0.09 0 1.97 3925 0.08 0.11 2.3n.d. = no data

TABLE 3 Comparative ELISA Titers between ParaChek ® and FUIDI Map ELISATests in Sera with Newly Developed Map Antibodies ParaChek ® Cow # Dec.13, 2006 Feb. 28, 2008 FUIDI#1 637 n.d. 0.06 3.1 1664 0 0.06 1.9 2139 00.02 1.6 2393 0 0.01 1.5 3111 0 0.03 2.0 3485 0 0.03 0.89 3523 0 0.011.1 3783 0 0.04 1.3 3790 0 0.02 2.0 3811 0 0.02 3.3 4259 n.d. 0.05 1.24n.d. = no data

TABLE 4 Correlation of FUIDI-positive Sera with Prior ParaChek ® ResultsParaChek ® Cow # Dec. 13, 2006 Feb. 28, 2008 FUIDI#1 FUIDI#1 Titer (2.5and greater) 128 0 0 2.6 637 n.d. 0.06 3.1 1011 0.08 2.38 2.5 2109 n.d.0 2.9 2761 n.d 0 3.2 2920 1.42 n.d 3.6 3107 0.2 0 3.3 3297 0.04 0 3.43387 n.d. 0.05 3.4 3536 n.d. 0 2.9 3811 0 0.02 3.3 3945 0 0.05 3.4 n.d.= no data

TABLE 5 Correlation of UFIDI-Positive and -Suspicious MAP ELISA Titerswith Any ParaChek ® Titer ParaChek ® Titer Cow # UFIDI ELISA#1 Dec. 13,2006 Feb. 28, 2008 UFIDI Titer (2.5 or greater)  128 2.6 0 0  637* 3.1 —0.06 1011 2.5 0.08 2.38 1663 2.9 0 0 2109 2.9 0 2674 3.3 — — 2761 3.2 02920 3.6 1.42 — 3107 3.3 0.2 0 3160 2.6 0 0 3297 3.4 0.04 0 3536 2.9 0 03702 2.5 0 0 3811 3.3 0 0.02 3945 3.4 0 0.05 3387 3.1 — 0 4320 2.6 — 0FUID#1 Titer (1.9-2.49)  480 2.3 n.d. 1004 2.1 0 2676 2.3 0 2891 2.0 03103 2.0 0 3111 2.0 0.03 3611 2.12 0 3621 2.1 0 3652 1.9 0 3728 2.1 0.080 3735 2.3 0 3739 1.97 0.09 0 3763 2.1 0 3790 2.0 0.02 3925 2.3 0.080.11 3953 2.1 0 4115 1.9 0 4194 2.1 0.02 4212 1.9 0 4314 2.1 0 4471 2.30 n.d. = no data

TABLE 6 Correlation of ParaChek ® and FUIDI Map ELISA Titers withPositive Fecal Cultures ParaChek ® ELISA FUIDI ELISA#1 Cow # 2008 2008Culture 1011 2.38 (positive) 2.5 (positive) ls 3160 0 2.16 (suspicious)ms 4156 0 1.3 (negative) ls 4237 0 1.4 (negative) ls 3394 0 1.6(negative) ms 4101 0 1.1 (negative) ms 4115 0 1.9 (suspicious) ms 41940.02 2.1 (suspicious) hs 4212 0 1.9 (suspicious) ms 4235 0 1.3(negative) ls 4237 0.01 1.3 (negative) ms ls = light shedder ms = mediumshedder hs = heavy shedder

1. The invention is a sequential and selective use of test results designed to identify animal with infection or disease due to Mycobaterium avium subspecies paratuberculosis (Map) and genomically related mycobacteria from which herd management decision can be made.
 2. The initial serological Map ELISA test used according to claim 1, wherein are Map ELISA tests which identify surface and intracellular antigens of Map and/or genomically related mycobacterium.
 3. When indicated by a suspicious or positive test value derived by Map ELISA test in claim 2, a second serological Map ELISA test whose antibody capturing ability is contingent upon Map or related mycobacterium lipoarabinomannan polysaccharides and/or membrane lipoproteins is employed to further analyze the significance of the data derived.
 4. The information derived from the test in claim 3 is used to create subcategories for analysis of significance: negative, low non-diagnostic titer, diagnostic titer as determined by the test's manufacturer.
 5. The information derived from the test in claim 3 for diagnostic category of negative requires no action unless the fecal culture demonstrates the presence of a large number of Map or related mycobacteria.
 6. The information derived from the test in claim 3 for diagnostic category of low non-diagnostic titer is deemed to indicate active infection and the guidelines recommend repeated test of the animal's serum using a Map ELISA lipoarabinomannan polysaccharide or lipoprotein based test in no less than 3 months.
 7. The information derived from the test in claim 3 for diagnostic category of positive forms the basis for recommending removal of the animal from the herd.
 8. The information derived from the test in claim 4 results in the creation of 3 categories of loss of titer, persistent comparable titer, and increasing titer.
 9. The information derived from the test in claim 4 for the category of loss of titers results in the conclusion that immune capture has been achieved and the recommendation that annual Map surveillance be continued and/or done after calving.
 10. The information derived from the test in claim 4 for the category of persistent, relatively comparable titer results in the conclusion of continue mycobacterium replication and the recommendation to continue test of serum, add testing milk using a Map PCR monthly and do fecal culturing.
 11. The information derived from the test in claim 10 from fecal culture results in 3 categories: negative, low shedder, high shedder.
 12. The information derived from the test in claim 10 for the category of low shedder results in conclusion of continued active mycobacterium replication and recommendations to either cull or repeated test of the animal's serum using a Map ELISA lipoarabinomannan polysaccharide or lipoprotein based test in no less than 3 months and testing milk using a Map PRC test monthly.
 13. The information derived from the test in claim 10 for the category of high shedder results in conclusion of continued active significant mycobacterium replication and recommendations to cull the animal from the herd.
 14. The information derived from the test in claim 4 for the category of diagnostic results in conclusion of significant active mycobacterium replication and recommendations to cull the animal from the herd.
 15. For animals meeting the criteria identified in claim 6,10,12, and prior to parturition, use of dietary supplementation which enhance the animal's immune system is strongly advocated. 